Myelin Metabolism and Metachromatic Leukodystrophy
Metachromatic leukodystrophy (MLD) is caused by an autosomal recessive genetic defect in the lysosomal enzyme Arylsulfatase A (ASA), resulting in a progressive breakdown of membranes of the myelin sheath (demyelination) and accumulation of galactosyl sulphatide (cerebroside sulphate) in the white matter of both the central nervous system (CNS) and the peripheral nervous system. In histologic preparations, galactosyl sulphatide forms spherical granular masses that stain metachromatically. Galactosyl sulphatide also accumulates within the kidney, gallbladder, and certain other visceral organs and is excreted in excessive amounts in the urine.
Multiple sulfatase deficiency (MSD) is a rare form of MLD that also includes features of mucopolysaccharidosis (MPS). MSD is characterised by a decreased activity of all known sulfatases. The clinical phenotype of MSD combines features of MLD with that of MPS as a result of the impaired lysosomal catabolism of sulphated glycolipids and glycosaminoglycans.
Galactosyl sulfatide is normally metabolised by the hydrolysis of 3-O-sulphate linkage to form galactocerebroside through the combined action of the lysosomal enzyme arylsulfatase A (EC 3.1.6.8) (Austin et al. Biochem J. 1964, 93, 15C-17C) and a sphingolipid activator protein called saposin B. A profound deficiency of arylsulfatase A occurs in all tissues from patients with the late infantile, juvenile, and adult forms of MLD (see below). In the following, the arylsulfatase A protein will be termed “ASA” and the saposin B will be termed “Sap-B”. A profound deficiency of ASA occurs in all tissues from patients with MLD.
ASA has been purified from a variety of sources including human liver, placenta, and urine. It is an acidic glucoprotein with a low isoelectric point. Above pH 6.5, the enzyme exists as a monomer with a molecular weight of approximately 100 kDa. ASA undergoes a pH-dependent polymerisation forming a dimer at pH 4.5. In human urine, the enzyme consists of two nonidentical subunits of 63 and 54 kDa. ASA purified from human liver, placenta, and fibroblasts also consist of two subunits of slightly different sizes varying between 55 and 64 kDa. As in the case of other lysosomal enzymes, ASA is synthesised on membrane-bound ribosomes as a glycosylated precursor. It then passes through the endoplasmic reticulum and Golgi, where its N-linked oligosaccharides are processed with the formation of phosphorylated and sulfated oligosaccharide of the complex type (Waheed A et al. Biochim Biophys Acta. 1985, 847, 53-61, Braulke T et al. Biochem Biophys Res Commun. 1987, 143, 178-185). In normal cultured fibroblasts, a precursor polypeptide of 62 kDa is produced, which translocates via mannose-6-phosphate receptor binding (Braulke T et al. J Biol Chem. 1990, 265, 6650-6655) to an acidic prelysosomal endosome (Kelly B M et al. Eur J Cell Biol. 1989, 48, 71-78).
The length (18 amino acids) of the human ASA signal peptide is based on the consensus sequence and a specific processing site for a signal sequence. Hence, from the deduced human ASA cDNA (EMBL GenBank accession numbers J04593 and X521151, see below) the cleavage of the signal peptide should be done in all cells after residue number 18 (Ala), resulting in the mature form of the human ASA. In the following, recombinant arylsulfatase A will be abbreviated rASA. the mature form of arylsulfatase A including the mature form of human ASA will be termed “mASA” and the mature recombinant human ASA will be termed “mrhASA”.
A protein modification has been identified in two eukaryotic sulfatases (ASA and arylsulfatase B (ASB)) and for one from the green alga Volvox carteri (Schmidt B et al. Cell. 1995, 82, 271-278, Selmer T et al. Eur J Biochem. 1996, 238, 341-345). This modification leads to the conversion of a cysteine residue, which is conserved among the known sulfatases, into a 2-amino-3-oxopropionic acid residue (Schmidt B et al. Cell. 1995, 82, 271-278). The novel amino acid derivative is also recognised as Cα-formylglycin (FGly). In ASA and ASB derived from MSD cells, the Cys-69 residue is retained. Consequently, it is proposed that the conversion of the Cys-69 to FGly-69 is required for generating catalytically active ASA and ASB, and that deficiency of this protein modification is the cause of MSD. Cys-69 is referred to the precursor ASA which has an 18 residue signal peptide. In the mASA the mentioned cysteine residue is Cys-51. Further investigations have shown that a linear sequence of 16 residues surrounding the Cys-51 in the mASA is sufficient to direct the conversion and that the protein modification occurs after or at a late stage of co-translational protein translocation into the endoplasmic reticulum when the polypeptide is not yet folded to its native structure (Dierks T et al. Proc Natl Acad Sci. 1997, 94, 11963-1196, Wittke, D. et al. (2004), Acta Neuropathol. (Berl.), 108, 261-271).
Multiple forms of ASA have been demonstrated on electrophoresis and isoelectric focusing of enzyme preparations from human urine, leukocytes, platelets, cultured fibroblasts and liver. Treatment with endoglycosidase H, sialidase, and alkaline phosphatase reduces the molecular size and complexity of the electrophoretic pattern, which suggests that much of the charge heterogeneity of ASA is due to variations in the carbohydrate content of the enzyme.
Clinical Manifestations of MLD
The central nervous system consists of the brain and the spinal cord, and can be divided into white and grey matter. The white matter consists of nerve cells, and in MLD the damage occurs primary in the nerve cells. When the nerve cells are damaged, they can no longer conduct nerve impulses to muscles, skin and internal organs.
In cases of MLD, there is a defect in ASA activity affecting myelin metabolism. Lack of this enzyme in patients with MLD leads the degradation of myelin and to dysfunction of the nerve cells. A concomitant accumulation of special types of fat in the nerve cells is also observed in MLD. Three forms of the disease can be distinguished according to the three forms of the age of onset: Late-infantile, juvenile and adult (after the age of 20 years). The course of the disease varies in the different types. The type occurring in early childhood is the commonest, progresses most rapidly, and leads to pronounced handicapping and death.
In the infantile form of MLD there are several stages of the disease. The first stage is characterised by slack muscles (hypotonia) of the arms and legs. Walking deteriorates and the child needs support to walk. The picture is often complicated by disturbances of balance (ataxia) and weakened muscle reflexes. In the second stage, about 1-1½ years after the onset, the child can no longer stand, but it can still sit. The previous slack muscles become spastic. The disturbance of balance gets worse, and pain in the arms and legs is commonly observed. The disease progresses to the third stage after additional 3-6 months where the child has increasing paralysis of all four limbs and can no longer sit. The child gradually needs help with everything, vision is impaired, and movements become difficult.
The juvenile type of MLD starts between the ages of five and ten years. The progression is similar to the infantile type, but slower. Emotional lability and impaired vision may be the first symptoms of the disease. In the adult form of MLD the symptoms arise in the age after 20 years after normal development. The symptoms include cognitive and behavioural abnormalities.
Incidence of MLD
In Norway, about one child with MLD is born every year, i.e. a frequency of about 1:50.000. Similar results have been obtained in northern Sweden where the birth incidence rate for late infantile MLD in this population can be calculated to be about 1 per 40.000. Only one patient with juvenile MLD was born in the mentioned region during the same period. This demonstrates that the juvenile form of MLD is much more rare than the infantile form.
Animal Model of MDL
ASA knockout mice develop a disease, which corresponds to MLD (Hess et al. 1996, Proc. Natl. Acad. Sci. U.S.A. 93, 14821-14826, Gieselmann, V. et al. 1989 J. Inherit. Metab. Dis., 21, 564-574, Gieselmann, V. et al. 2003, Acta Paediatr. Suppl., 92, 74-79). Thus, they display storage deposits with a distribution and ultrastructure which is virtually identical to those in patients. The mice develop neurologic symptoms reminiscent of the human disease comprising gait disturbancies, reduced motor coordination abilities and hyperactivity (Hess et al. 1996, Proc. Natl. Acad. Sci. U.S.A. 93, 14821-14826, D'Hooge, R. et al. 2001, Brain Res., 907, 35-43, Matzner, U. et al. 2002, Gene Ther., 9, 53-63). The symptoms become apparent at around one year of age, but they do not reduce the life expectancy of the mice. The mild phenotype has been explained by the lack of widespread demyelination (Hess et al. 1996, Proc. Natl. Acad. Sci. U.S.A. 93, 14821-14826, Coenen, R. et al. 2001, Acta Neuropathol. (Berl.)., 101, 491-498, Wittke, D. et al. 2004, Acta Neuropathol. (Berl.), 108, 261-271). The limited demyelination in mice can be attributed to the short life span, which does not allow for the development of cellular dysfunctions, causative for demyelination. The ASA knock out mice therefore represent an appropriate animal model particularly for investigating therapeutic interventions in an early stage of the human disease.
Existing Diagnosis of MLD
In order to diagnose MLD, examination of spinal fluid, urine, various blood tests, and analysis of the ASA activity can be carried out. Deficiency of ASA activity in material from patients with MLD (e.g. peripheral leukocytes and cultured skin fibroblasts) can be investigated. Analysis of the urine from patients with MLD can indicate a defect at the level of myelin metabolism but this is a less reliable source for diagnostic assays because the urinary enzyme level is normally highly variable. Excessive amounts of sulpatide excreted in the urine and metachromatic granules in the urinary sediment are observed. Furthermore, normal x-rays and computer tomography (CT) of the head may be carried out. Prenatal diagnosis appears to be possible by measuring ASA activity in cultured cells from amniotic fluid or chorionic villus cells. Cerebroside sulfate loading of such cells can also be used and is the method of choice if the pseudodeficiency gene is also present in the family.
Existing Treatment of MLD
There are relatively few treatment options for MLD. Bone Marrow Transplantation (BMT) has been used in the treatment of more than 20 patients with MLD (for instance Bayever E et al. Lancet 1985, 2, 471-473), and it appears that BMT slows the progression of symptoms, but benefits of the treatment are not seen for several months. In most late infantile patients, symptoms are progressing rapidly by the time of diagnosis, and the risks of the procedure tend to outweigh the possible benefits. In instances in which the diagnosis can be made presymtomatically and a well-matched donor is available, BMT may be a reasonable approach. Moreover, reported results suggest that BMT is efficacious only in MLD patients with high residual activity or when performed in presymptomatic stages in the late infantile form probably because of the rapid progression of the disease. The perspective of using bone marrow transplantation is further limited by the fact that it only reduces symptoms in the central nervous system and that supplementary treatment is required in order to alleviate symptoms in the peripheral nervous system.
Cell culture models suggest that cysteine protease inhibitor treatment (von Figura K et al. Am J Hum Genet 1986, 39, 371-382), thiosulfate treatment (Eto Y et al. Biochem Biophys Res Commun 1982, 106, 429-434), enzyme replacement (Porter M T Science 1971, 172 (989), 1263-1265), and gene replacement therapies (Sangalli A et al. Hum Gene Ther 1998, 9, 2111-2119) could be effective. Several possible gene therapy approaches have been suggested.
In one of these approaches an implanted polymer-encapsulated xenogenic transduced cell line secreting the ASA enzyme is used. This approach has previously been used for the treatment of other neurological disorders such as Amyotrophic Lateral Sclerosis and Parkinson disease. A cathetered devise, containing around 106 genetically modified cells surrounded by a semipermeable membrane, is suggested to be implanted in the ventricular space, providing slow continuous release of ASA directly in cerebral spinal fluid. For this gene transfer technique C2C12 mouse myoblast cells are used (Degion et al. Hum Gene Ther 1996, 7, 2135-2146). The semipermeable membrane prevents immunologic rejection of the cells and interposes a physical barrier between cells and host. Moreover, the device and the cells may be retrieved in the event of side effect due to the ASA administration.
In another approach, ASA genes are directly delivered into the brain by the use of recombinant adenovirus (Ohashi et al. Acta Paediatr Jpn. 1996, 38, 193-201). It was shown that the recombinant adenovirus (Adex1SRLacZ) was able to transduce the oligodendrocytes very efficiently. Despite the fact that gene therapy have led to satisfactory increases in tissue enzyme levels, the success of this approach appears limited, as studies have revealed no significant decline in the sulfatide levels in response to the increased enzyme levels in important tissues such as the kidney. The disappointing results may be caused by insufficient translocation of arylsulfatase A to the lysosomes.
Conventional Enzyme Replacement Therapy based on systemic infusion of arylsulfatase A would clearly provide cost-efficient treatment of MLD with little inconvenience and low risk of complications to the patients. As opposed to gene therapy, enzyme replacement therapy would also not raise any ethical questions. The application of enzyme replacement therapy in the treatment of MLD has, however, been hampered by the difficulties in preparing large amounts of arylsulfatase A with sufficient specific activity and at the quality required for clinical applications. Furthermore, enzyme replacement therapy is traditionally considered efficient only in reducing sulfatide levels in the peripheral nervous system, since arylsulfatase due to its size is unlikely to access the central nervous system.